The characterization of cellular gene expression finds application in a variety of disciplines, such as in the analysis of differential expression between different tissue types, different stages of cellular growth or between normal and diseased states. Recently, changes in gene expression have also been used to assess the activity of new drug candidates and to identify new targets for drug development. The latter objective is accomplished by correlating the expression of a gene or genes known to be affected by a particular drug with the expression profile of other genes of unknown function when exposed to that same drug; genes of unknown function that exhibit the same pattern of regulation, or signature, in response to the drug are likely to represent novel targets for pharmaceutical development. One particularly useful method of assaying gene expression at the level of transcription employs DNA microarrays (Ramsay, Nature Biotechnol. 16: 40–44, 1998; Marshall and Hodgson, Nature Biotechnol. 16: 27–31, 1998; Lashkari et al., Proc. Natl. Acad. Sci. (USA) 94: 130–157, 1997; DeRisi et al., Science 278: 680–6, 1997).
A number of methods for the amplification of nucleic acids have been described. Such methods include the “polymerase chain reaction” (PCR) (Mullis et al., U.S. Pat. No. 4,683,195), and a number of transcription-based amplification methods (Malek et al., U.S. Pat. No. 5,130,238; Kacian and Fultz, U.S. Pat. No. 5,399,491; Burg et al., U.S. Pat. No. 5,437,990). Each of these methods uses primer-dependent nucleic acid synthesis to generate a DNA or RNA product, which serves as a template for subsequent rounds of primer-dependent nucleic acid synthesis. Each process uses (at least) two primer sequences complementary to different strands of a desired nucleic acid sequence and results in an exponential increase in the number of copies of the target sequence. These amplification methods can provide enormous amplification (up to billion-fold). However, these methods have limitations that make them not amenable for gene expression monitoring applications. First, each process results in the specific amplification of only the sequences that are bounded by the primer binding sites. Second, exponential amplification can introduce significant changes in the relative amounts of specific target species—small differences in the yields of specific products (for example, due to differences in primer binding efficiencies or enzyme processivity) become amplified with every subsequent round of synthesis.
Amplification methods that utilize a primer containing a RNA polymerase promoter sequence (“promoter-primer”) are amenable to the amplification of heterogeneous mRNA populations. The vast majority of mRNAs carry a homopolymer of 20–250 adenosine residues on their 3′ ends (the poly-A tail), and the use of poly-dT primers for cDNA synthesis is a fundamental tool of molecular biology. “Single-primer amplification” protocols have been reported (see e.g., Kacian et al., U.S. Pat. No. 5,554,516; Van Gelder et al., U.S. Pat. No. 5,716,785). The methods reported in these patents utilize a single promoter-primer containing a RNA polymerase promoter sequence and a sequence complementary to the 3′-end of the desired nucleic acid target sequence(s). In both methods, the promoter-primer is added under conditions in which it hybridizes to the target sequence(s) and is converted to a substrate for RNA polymerase. In both methods, the substrate intermediate is recognized by RNA polymerase, which produces multiple copies of RNA complementary to the target sequence(s) (“antisense RNA”). Each method uses, or could be adapted to use, a primer containing poly-dT for amplification of heterogeneous mRNA populations.
Amplification methods that proceed linearly during the course of the amplification reaction are less likely to introduce bias in the relative levels of different mRNAs than those that proceed exponentially. In the method described in Kacian et al., U.S. Pat. No. 5,554,516, the amplification reaction contains a nucleic acid target sequence, a promoter-primer, a RNA polymerase, a reverse transcriptase, and reagent and buffer conditions sufficient to allow amplification. The amplification proceeds in a single tube under conditions of constant temperature and ionic strength. Under these conditions, the antisense RNA products of the reaction can serve as substrates for further amplification by non-specific priming and extension by the RNA-dependent DNA polymerase activity of reverse transcriptase. As such, the amplification described in U.S. Pat. No. 5,554,516 proceeds exponentially. In contrast, in specific examples described in Van Gelder et al., U.S. Pat. No. 5,716,785, cDNA synthesis and transcription occur in separation reactions separated by phenol/chloroform extraction and ethanol precipitation (or dialysis), which may incidentally allow for the amplification to proceed linearly since the RNA products cannot serve as substrates for further amplification.
The method described in U.S. Pat. No. 5,716,785 has been used to amplify cellular mRNA for gene expression monitoring (for example, R. N. Van Gelder et al. (1990), Proc. Natl. Acad. Sci. USA 87, 1663; D. J. Lockhart et al. (1996), Nature Biotechnol. 14, 1675). However, this procedure is not readily amenable to high throughput processing. In preferred embodiments of the method described in U.S. Pat. No. 5,716,785, poly-A mRNA is primed with a promoter-primer containing poly-dT and converted into double-stranded cDNA using a method described by Gubler and Hoffman (U. Gubler and B. J. Hoffman (1983), Gene 25, 263–269) and popularized by commercially available kits for cDNA synthesis. Using this method for cDNA synthesis, first strand synthesis is performed using reverse transcriptase and second strand cDNA is synthesized using RNaseH and DNA polymerase I. After phenol/chloroform extraction and dialysis, double-stranded cDNA is transcribed by RNA polymerase to yield antisense RNA product. The phenol/chloroform extractions and buffer exchanges required in this procedure are labor intensive, and are not readily amenable to robotic handling.
A method of linear amplification of mRNA into antisense RNA has been recently developed, U.S. Pat. No. 6,132,997 issued to Shannon (“Shannon”), which is incorporated by reference in its entirety for all purposes. Shannon does not require a reverse transcriptase separation step and is therefore readily amenable to high throughput processing. Shannon discloses a method in which mRNA is converted to cDNA (particularly double-stranded cDNA) using a promoter-primer having a poly-dT primer site linked to a promoter sequence so that the resulting cDNA is recognized by a RNA polymerase. The resultant cDNA is then transcribed into RNA (particularly antisense RNA) in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during the transcription step.
A significant drawback of the Shannon method, however, is that it produces a 3′ bias in the amplification of mRNA. Sequences that are more than 1000 bp from the 3′ end to which the primer has hybridized are underamplified with respect to sequences that are less than 1000 bp from the 3′ end, i.e., the sequences that are more than 1000 bp from the 3′ end are amplified in less than linear amounts.
Thus there exists a need in the art for an improved method of linear amplification of mRNA that is amenable to high throughput processing, that produces little or no 3′ bias, that improves the ability to detect the 5′ ends of mRNA, and therefore achieves good representation of both the 3′ and 5′ regions of an original mRNA in the amplified complementary RNA (cRNA).